This study was conducted to find out the optimal solvent extraction method [Distilled water (DW), 70% ethanol, 99% ethanol] of mushrooms, including Pleurotus ostreatus (Jacq.) Que, Pleurotus eryngii and Flammulina velutipes and improve their usability as natural antioxidants. To analyze antioxidant activities in each mushroom, total polyphenol, flavonoid contents, 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2?-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) cation radical (ABTS+) and fluorescence recovery after photobleaching (FRAP) were measured. All mushrooms showed the highest total polyphenol contents in DW mushroom extract (p<0.001). Total flavonoid contents were the highest in P. eryngii and F. velutipes DW and 70% ethanol mushroom extracts (p<0.05). All mushrooms showed the highest activities using DPPH and FRAP assays in the DW extraction method (p<0.001). P. ostreatus (Jacq.) Que and P. eryngii showed the highest ABTS+ radical scavenging activity in the DW extraction method, and F. velutipes showed the highest activity in the 70% ethanol extraction method (p<0.001). As a result of comparing IC50 values of DPPH and ABTS+ radicals and FRAP EC50 values, the DW P. ostreatus (Jacq.) Que extract showed high antioxidant activities (p<0.001). Pearson's correlation between total polyphenol contents and antioxidant activities showed a positive correlation in all mushrooms (p<0.01). Therefore, extraction of the mushrooms with DW can enhance the extraction of effective bioactive substances and antioxidant activity.